Aminoacyl-tRNA synthetases and their inhibitors as a novel family of antibiotics

The emergence of multidrug-resistant strains of pathogenic microorganisms and the slow progress in new antibiotic development has led in recent years to a resurgence of infectious diseases that threaten the well-being of humans. The result of many microorganisms becoming immune to major antibiotics means that fighting off infection by these pathogens is more difficult. The best strategy to get around drug resistance is to discover new drug targets, taking advantage of the abundant information that was recently obtained from genomic and proteomic research, and explore them for drug development. In this regard, aminoacyl-tRNA synthetases (ARSs) provide a promising platform to develop novel antibiotics that show no cross-resistance to other classical antibiotics. During the last few years there has been a comprehensive attempt to find the compounds that can specifically target ARSs and inhibit bacterial growth. In this review, the current status in the development of ARS inhibitors will be briefly summarized, based on their chemical structures and working mechanisms.     

Innovative approaches to novel antibacterial drug discovery

Increasing antibiotic resistance in microorganisms and new emerging pathogens have become a major problem in our society. Rising to satisfy this urgent medical need is a recent confluence of powerful new drug discovery technologies: combinatorial chemistry; sequence and functional genomic analysis; and novel methods of high-throughput screening. The combination of these technologies will bring to bear untapped power in the search for new antimicrobials.     

Functional genomics and target validation approaches using antisense oligonucleotide technology

The recent increase in the amount and rate of accumulation of genomic information has created new challenges for the pharmaceutical industry. These include how best to rapidly and efficiently identify key genes responsible for complex disease phenotypes and how to use this information to develop new and specific classes of drugs. Antisense technology offers a powerful approach to identify novel cellular networks and signaling "cassettes" and provides a method to validate genes in vivo as attractive drug targets.     

Role of xenobiotic transporters in bacterial drug resistance and virulence

Since the discovery of antibiotic therapeutics, the battles between humans and infectious diseases have never been stopped. Humans always face the appearance of a new bacterial drug-resistant strain followed by new antibiotic development. However, as the genome sequences of infectious bacteria have been gradually determined, a completely new approach has opened. This approach can analyze the entire gene resources of bacterial drug resistance. Through analysis, it may be possible to discover the underlying mechanism of drug resistance that will appear in the future. In this review article, we will first introduce the method to analyze all the xenobiotic transporter genes by using the genomic information. Next, we will discuss the regulation of xenobiotic transporter gene expression through the two-component signal transduction system, the principal environmental sensing and response system in bacteria. Furthermore, we will also introduce the virulence roles of xenobiotic transporters, which is an ongoing research area.     

Genomic characterization of antibiotic resistance-encoding genes in clinical isolates of Vibrio cholerae non-O1/non-O139 strains from Kolkata,India: generation of novel types of genomic islands containing plural antibiotic resistance genes

Non-O1/non-O139 nontoxigenic Vibrio cholerae associated with cholera-like diarrhea has been reported in Kolkata, India. However, the property involved in the pathogenicity of these strains has remained unclear. The character of 25 non-O1/non-O139 nontoxigenic V. cholerae isolated during 8 years from 2007 to 2014 in Kolkata was examined. Determination of the serogroup showed that the serogroups O6, O10, O35, O36, O39, and O70 were represented by two strains in each serogroup, and the remaining isolates belonged to different serogroups. To clarify the character of antibiotic resistance of these isolates, an antibiotic resistance test and the gene analysis were performed. According to antimicrobial drug susceptibility testing, 13 strains were classified as drug resistant. Among them, 10 strains were quinolone resistant and 6 of the 13 strains were resistant to more than three antibiotics. To define the genetic background of the antibiotic character of these strains, whole-genome sequences of these strains were determined. From the analysis of these sequences, it becomes clear that all quinolone resistance isolates have mutations in quinolone resistance-determining regions. Further research on the genome sequence showed that four strains possess Class 1 integrons in their genomes, and that three of the four integrons are found to be located in their genomic islands. These genomic islands are novel types. This indicates that various integrons containing drug resistance genes are spreading among V. cholerae non-O1/non-O139 strains through the action of newly generated genomic islands.     

ACX3, a novel medium-chain acyl-coenzyme A oxidase from Arabidopsis

In a database search for homologs of acyl-coenzyme A oxidases (ACX) in Arabidopsis, we identified a partial genomic sequence encoding an apparently novel member of this gene family. Using this sequence information we then isolated the corresponding full-length cDNA from etiolated Arabidopsis cotyledons and have characterized the encoded recombinant protein. The polypeptide contains 675 amino acids. The 34 residues at the amino terminus have sequence similarity to the peroxisomal targeting signal 2 of glyoxysomal proteins, including the R-[I/Q/L]-X5-HL-XL-X15-22-C consensus sequence, suggesting a possible microsomal localization. Affinity purification of the encoded recombinant protein expressed in Escherichia coli followed by enzymatic assay, showed that this enzyme is active on C8:0- to C14:0-coenzyme A with maximal activity on C12:0-coenzyme A, indicating that it has medium-chain-specific activity. These data indicate that the protein reported here is different from previously characterized classes of ACX1, ACX2, and short-chain ACX (SACX), both in sequence and substrate chain-length specificity profile. We therefore, designate this new gene AtACX3. The temporal and spatial expression patterns of AtACX3 during development and in various tissues were similar to those of the AtSACX and other genes expressed in glyoxysomes. Currently available database information indicates that AtACX3 is present as a single copy gene.     

Microbial genomics: rhetoric or reality?

The availability of complete genome sequences of many bacterial species is facilitating numerous computational approaches for understanding bacterial genomes. One of the major incentives behind the genome sequencing of many pathogenic bacteria is the desire to better understand their diversity and to develop new approaches for controlling human diseases caused by these microorganisms. This task has become even more urgent with the rapid evolution of antibiotic resistance among many bacterial pathogens. Novel drug targets are required in order to design new antimicrobials against antibiotic-resistant pathogens. The complete genome sequences of an ever increasing number of pathogenic microbes constitute an invaluable resource and provide lead information on potential drug targets. This review focuses on in silico analyses of microbial genomes, their host-specific adaptations, with specific reference to genome architecture, design, evolution, and trends in computational identification of microbial drug targets. These trends underscore the utility of genomic data for systematic in silico drug target identification in the post-genomic era.     

Mining nematode genome data for novel drug targets

Expressed sequence tag projects have currently produced over 400 000 partial gene sequences from more than 30 nematode species and the full genomic sequences of selected nematodes are being determined. In addition, functional analyses in the model nematode Caenorhabditis elegans have addressed the role of almost all genes predicted by the genome sequence. This recent explosion in the amount of available nematode DNA sequences, coupled with new gene function data, provides an unprecedented opportunity to identify pre-validated drug targets through efficient mining of nematode genomic databases. This article describes the various information sources available and strategies that can expedite this process.     

Functional genomics: identifying drug targets for parasitic diseases

The genomic sequences of parasitic diseases are rapidly becoming available and, recently, the full sequence of Plasmodium falciparum has been published. Much has been promised from this genomic revolution including the identification of new drug targets and novel chemotherapeutic treatments for the control of parasitic diseases. The challenge to use this information efficiently will require functional genomics tools such as bioinformatics, microarrays, proteomics and chemical genomics to identify potential drug targets, and to allow the development of optimized lead compounds. The information generated from these tools will provide a crucial link from genomic analysis to drug discovery.     

Self-medication and antibiotic resistance: Crisis,current challenges,and prevention

The present study aims to explore the crisis of antibiotic resistance and discover more about the current challenges related to self-medication. The current challenges related to antibiotic resistance are unique and differ from the challenges of the past since new bacterial pathogens are involved and continue to evolve. Strains with resistance to multiple antibiotic classes have emerged which the discovery of new antibiotics has failed to match. The consequences of antibiotic resistance are grave with mortality and morbidity continually on the rise. This paper also highlights the possible interventions that can be effective at the micro or individual level in the prevention of development of antibiotic resistance.     

Metabolic pathway analysis in trypanosomes and malaria parasites

Identification of novel drug targets is required for the development of new classes of drugs to overcome drug resistance and replace less efficacious treatments. In theory, knowledge of the entire genome of a pathogen identifies every potential drug target in any given microbe. In practice, the sheer complexity and the inadequate or inaccurate annotation of genomic information makes target identification and selection somewhat more difficult. Analysis of metabolic pathways provides a useful conceptual framework for the identification of potential drug targets and also for improving our understanding of microbial responses to nutritional, chemical and other environmental stresses. A number of metabolic databases are available as tools for such analyses. The strengths and weaknesses of this approach are discussed.     

Drug discovery for malaria: a very challenging and timely endeavor

The prevalence of resistance to known antimalarial drugs has resulted in the expansion of antimalarial drug discovery efforts. Academic and nonprofit institutions are partnering with the pharmaceutical industry to develop new antimalarial drugs. Several new antimalarial agents are undergoing clinical trials, mainly those resurrected from previous antimalarial drug discovery programs. Novel antimalarials are being advanced through the drug development process, of course, with the anticipated high failure rate typical of drug discovery. Many of these are summarized in this review. Mechanisms for funding antimalarial drug discovery and genomic information to aid drug target selection have never been better. It remains to be seen whether ongoing efforts will be sufficient for reducing malaria burden in the developing world.     

Antibiotic resistance in bacteria and its future for novel antibiotic development

Since the first introduction of the sulfa drugs and penicillin into clinical use, large numbers of antibiotics have been developed and hence contributed to human health. But extensive use of antibiotics has raised a serious public health problem due to multiantibiotic resistant bacterial pathogens that inevitably develop resistance to every new drug launched in the clinic. Consequently, there is a pressing need to develop new antibiotics to keep pace with bacterial resistance. Recent advances in microbial genomics and X-ray crystallography provide opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics respectively. To prevent and control infectious diseases caused by multiantibiotic resistant bacteria, we need to understand more about the molecular aspects of the pathogens' physiology and to pursue ways to prolong the life of precious antibiotics.     

Lipopolysaccharide as an antibiotic target

Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii are amongst the highest priority drug-resistant pathogens, for which new antibiotics are urgently needed. Whilst antibiotic drug development is inherently challenging, this is particularly true for Gram-negative bacteria due to the presence of the outer membrane, a highly selective permeability barrier that prevents the ingress of several classes of antibiotic. This selectivity is largely due to an outer leaflet composed of the glycolipid lipopolysaccharide (LPS), which is essential for the viability of almost all Gram-negative bacteria. This essentiality, coupled with the conservation of the synthetic pathway across species and recent breakthroughs in our understanding of transport and membrane homeostasis has made LPS an attractive target for novel antibiotic drug development. Several different targets have been explored and small molecules developed that show promising activity in vitro. However, these endeavours have met limited success in clinical testing and the polymyxins, discovered more than 70 years ago, remain the only LPS-targeting drugs to enter the clinic thus far. In this review, we will discuss efforts to develop therapeutic inhibitors of LPS synthesis and transport and the reasons for limited success, and explore new developments in understanding polymyxin mode of action and the identification of new analogues with reduced toxicity and enhanced activity.     

Genomic medicine: bringing biomarkers to clinical medicine

An important by-product of sequencing the human genome has been the development of a novel 'toolbox' for biomarker discovery and development. Genomic medicine is an emerging discipline in the genome sciences that integrates these tools to interrogate genomic variation in well-defined populations in order to develop predictors of disease susceptibility, progression and drug response. Several important classes of biomarkers result from these analyses which, when translated to clinical medicine and drug development, will have an important impact on human health and disease. This review highlights both the opportunities and challenges in bringing biomarkers into clinical medicine.     

Proteome of a methicillin-resistant Staphylococcus aureus clinical strain of sequence type ST398

Proteomics is a powerful tool to analyze the differences in gene expression of bacterial strains. Staphylococcus aureus has long been recognized as an important pathogen in human disease. In order to investigate this pathogen, the proteome of a clinical methicillin-resistant S. aureus (MRSA) strain of the sequence type ST398 was determined using 2-DE. Using 2-DE we obtained a total of 105 spots the MRSA strain. Furthermore in correlation with bioinformatic databases, they allowed accurate identification and characterization of proteins, resulting in 227 identified proteins. There were found proteins related to basic function of the cell, but also proteins related to virulence like catalase, specific of S. aureus species, and proteins related to antibiotic resistance. Proteins associated with antibiotic resistance or virulence factors are related to genomic databases. The most abundant classes identified involved glycolysis, energy production, one-carbon metabolism, and oxidation-reduction process, all of which reflect an active metabolism. These results highlight the importance of proteomics to deepen in the knowledge of protein expression of MRSA strain of the lineage ST398, microorganism with diverse and important resistance mechanisms. With this proteome map we have an essential tool for a better understanding of this pathogen and providing new data for protein databases. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

The problem of antibiotic resistant bacteria. The important role of environmentally responsive mutagenesis, its relevance to a new paradigm that may allow a solution

The frequent creation of antibiotic resistant bacteria through mutation poses a severe medical problem. As a way towards solving the problem, mutations conferring antibiotic sensitivity could be induced in such bacteria at very high frequency through the controlled process of environmentally responsive mutagenesis, an adaptively responsive mutator process. Such induction, as a necessary developmental stage towards global adaptation, would be enabled by patterns of mild stresses. The stresses and their patterns would be elucidated through in vitro experiments and clinical trials. The effective application of this ordered process, whose detection was allowed through a change in experimental design, requires a new and more comprehensive paradigm of mutagenesis than the one currently applied. This would be a new paradigm that has holistic and developmental features. Yet, the effective and ongoing emergence of such a paradigm will depend on experimental setups that will enable or provide, unlike previous experimental arrangements, new insights as to the developmental connections between innerly controlled mutagenesis and its environments. This should demonstrate controlled ways to generate and make manifest those genomic changes that, as developmental stages, give rise to antibiotic sensitivity in antibiotic resistant bacteria. The ultimate consequence of such a developed paradigm of mutagenesis may very well be a major and benign medical and biotechnical revolution.     

Comparative genomic analysis of Acinetobacter baumannii clinical isolates reveals extensive genomic variation and diverse antibiotic resistance determinants

Background

Acinetobacter baumannii is an important nosocomial pathogen that poses a serious health threat to immune-compromised patients. Due to its rapid ability to develop multidrug resistance (MDR), A. baumannii has increasingly become a focus of attention worldwide. To better understand the genetic variation and antibiotic resistance mechanisms of this bacterium at the genomic level, we reported high-quality draft genome sequences of 8 clinical isolates with various sequence types and drug susceptibility profiles.

Results

We sequenced 7 MDR and 1 drug-sensitive clinical A. baumannii isolates and performed comparative genomic analysis of these draft genomes with 16 A. baumannii complete genomes from GenBank. We found a high degree of variation in A. baumannii, including single nucleotide polymorphisms (SNPs) and large DNA fragment variations in the AbaR-like resistance island (RI) regions, the prophage and the type VI secretion system (T6SS). In addition, we found several new AbaR-like RI regions with highly variable structures in our MDR strains. Interestingly, we found a novel genomic island (designated as GI_BJ4) in the drug-sensitive strain BJ4 carrying metal resistance genes instead of antibiotic resistance genes inserted into the position where AbaR-like RIs commonly reside in other A. baumannii strains. Furthermore, we showed that diverse antibiotic resistance determinants are present outside the RIs in A. baumannii, including antibiotic resistance-gene bearing integrons, the bla_OXA-23-containing transposon Tn2009, and chromosomal intrinsic antibiotic resistance genes.

Conclusions

Our comparative genomic analysis revealed that extensive genomic variation exists in the A. baumannii genome. Transposons, genomic islands and point mutations are the main contributors to the plasticity of the A. baumannii genome and play critical roles in facilitating the development of antibiotic resistance in the clinical isolates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1163) contains supplementary material, which is available to authorized users.     

Genetic Architecture of Intrinsic Antibiotic Susceptibility

Background

Antibiotic exposure rapidly selects for more resistant bacterial strains, and both a drug''s chemical structure and a bacterium''s cellular network affect the types of mutations acquired.

Methodology/Principal Findings

To better characterize the genetic determinants of antibiotic susceptibility, we exposed a transposon-mutagenized library of Escherichia coli to each of 17 antibiotics that encompass a wide range of drug classes and mechanisms of action. Propagating the library for multiple generations with drug concentrations that moderately inhibited the growth of the isogenic parental strain caused the abundance of strains with even minor fitness advantages or disadvantages to change measurably and reproducibly. Using a microarray-based genetic footprinting strategy, we then determined the quantitative contribution of each gene to E. coli''s intrinsic antibiotic susceptibility. We found both loci whose removal increased general antibiotic tolerance as well as pathways whose down-regulation increased tolerance to specific drugs and drug classes. The beneficial mutations identified span multiple pathways, and we identified pairs of mutations that individually provide only minor decreases in antibiotic susceptibility but that combine to provide higher tolerance.

Conclusions/Significance

Our results illustrate that a wide-range of mutations can modulate the activity of many cellular resistance processes and demonstrate that E. coli has a large mutational target size for increasing antibiotic tolerance. Furthermore, the work suggests that clinical levels of antibiotic resistance might develop through the sequential accumulation of chromosomal mutations of small individual effect.